Protein localization, activity, and degradation are often controlled by post-translational modifications. One such modification is the covalent attachment of the small protein, ubiquitin. The work proposed here focuses on mechanistic aspects of protein ubiquitination. Ubiquitin is a highly conserved protein, with greater than 90% identity between ubiquitins of different species. The nematode, Caenorhabditis elegans, will be used as a model system for understanding the ubiquitination pathway. C. elegans provides an excellent system for this type of study because the genome is sequenced and annotated, a collection of ORF clones is available, a large database of known mutant phenotypes exists and many genetic and cell biological tools are available for studying cellular pathways. Since ubiquitination has been implicated in diseases such as breast cancer, Parkinson's disease, and Cystic Fibrosis it is important to have an understanding of the basic mechanisms of ubiquitination. Ubiquitination is typically achieved via a three-step pathway utilizing the E1 ubiquitin activating enzyme (E1), the E2 ubiquitin conjugating enzymes (Ubc), and the E3 ubiquitin ligases (E3). Interactions among the Ubcs and E3s are important for selecting which target proteins will be modified and for determining the nature of ubiquitination on the target protein. In addition, some Ubcs are known to associate with Ubc variants (Uev). Our preliminary studies on the ubiquitination pathway in C. elegans have included two-hybrid interaction studies and RNAi analysis of the Ubcs and E3s. In addition, an in vitro system for studying ubiquitination by C. elegans proteins has been developed. These studies will be extended via the following four specific aims. First, a yeast two-hybrid screen will used to test for interactions among the C. elegans Ubcs and Uevs. Identified interactions will be followed up by in vitro ubiquitination assays. Second, mutant analysis of Ubcs will be continued using varied RNAi conditions and available mutant alleles. Third, potential Ubc-E3 interactions will be identified using a two-hybrid screen. Again, these interactions will be tested using the in vitro ubiquitination assay. Fourth, the two interacting pairs, UBC-13 with UBC-1 and UBC-13 with UEV-1, will be studied in vitro to determine the nature of those interactions and how they affect E3 interaction and ubiquitin chain formation.